A methylotrophic strain can grow with methanol as a carbon and energy source. This is attributable to the fact that it has a gene encoding alcohol oxydase (EC1. 1. 3. B, hereinafter also referred to as AOX) which is an enzyme catalyzing a first reaction in the metabolism of methanol, namely, oxidation of methanol into formaldehyde.
A methylotrophic strain can metabolize methanol by the action of the AOX it produces even in a medium containing methanol as a sole carbon source and can grow on the metabolite of methanol as a carbon source.
Pichia pastoris is one of the methylotrophic yeasts and has two kinds of AOX genes, AOX1 gene and AOX2 gene, each having a promoter, a structural gene and a terminator in their chromosomes. Their structural genes have high homology with each other and code for AOXs having similar specific activities. As regards their promoters, however, they are known to have remarkably different transcription activities (AOX1 promoter&gt;AOX2 promoter) and AOX actually produced is mostly derived from the AOX1 gene [Molecular and Cellular Biology, Vol. 9, 1316 (1989)].
In recent years, methods for producing heterologous proteins by using AOX genes of a Pichia yeast have been studied [Yeast, 5, 167-177 (1989), U.S. Pat. No. 4,929,555, EP-A-344459, EP-A-347928]. A strain comprising a gene encoding a desired heterologous protein inserted in the place of a structural gene at the downstream from an AOX1 promoter having high promoter activity is superior in transcription efficiency, whereas the growth thereof in a medium containing methanol as a carbon source is poor due to a very small amount of produced AOX (which permits methanol utilization) by an AOX2 gene alone having poor expression activity, and an extended culture is necessary for producing a heterologous protein.
It has now been found that the mutation of the AOX2 promoter of the strain results in an enhanced promoter activity to afford an increased production of AOX by the AOX2 gene. Thus, the mutation of the AOX2 promoter has made it possible for the strain to grow well in a medium containing methanol.
Together, however, it has been found that the AOX promoters of the strain are subject to catabolite repression caused by glucose in a medium. That is, the transcription activity of AOX promoters is repressed in the presence of glucose in a medium, thus resulting in failure to express an AOX gene and a heterologous protein gene which are under the control of the above-mentioned promoters. Although the strain synthesizes its constitutive protein and can grow on glucose as a carbon source, it does not produce AOX and heterologous protein. The same applies to a medium containing both methanol and glucose. In this case, it is not until glucose has been completely consumed that AOX promoters begin to act to produce AOX and a heterologous protein.
Alternatively, the aforementioned strain undesirably requires an extended time for producing a heterologous protein, which is unbeneficial for efficient production of a desired heterologous protein.